The identification of potential new drug targets is important for the treatment of T. gondii. The PI has characterized cystein proteinase activity in tachyzoites of T. gondii, cloned a Toxoplasma cysteine proteinase gene and shown that potent new cystein proteinase inhibitors inhibit intracellular multiplication. The applicant will have access to the newest generation of cystein proteinase inhibitors as well as cores for expression of recombinant proteinases, computer modeling crystallography. They will test the hypothesis that cysteine proteinases play a critical role in the pathogenesis of toxoplasmosis and may be a novel drug target. Their first specific aim is to characterize the cysteine proteinases of T. gondii by expressing the recombinant enzyme comparing the recombinant and native enzymes identifying other potential cysteine proteinase genes and their gene products and producing specific antibody to the proteinase. The second aim is to evaluate the role of the proteinases in the pathogenesis of toxoplasmosis by localizing the proteinase within the parasite, evaluating the release of cystine proteinases and their role in peptide degradation and evaluating the effect of specific cysteine proteinase inhibitors on parasite survival and identify their site of action. The third aim is to determine the effect of blocking cysteine proteinases in vivo in mouse models of toxoplasmosis by blocking acute infection in mice with specific inhibitors and comparing the outcome of infection with parasites lacking the cys proteinase gene.